Prostate cancer is the second-leading cause of the cancer-related death in men in the United States. We have enriched prostate stem cell activity using the Sca-1 surface antigen and demonstrated that cells with enriched stem cell activity serve as a target for tumor initiation. Our long-term goals are to fully characterize the identity of stem cells and cancer-initiating cells in prostate. This study will provide general insights for the studies on stem cell biology and cancer biology. Our specific aims are: (1) Fully characterize the prostatic stem cells. (A) We will investigate whether stem cells reside in a specific prostatic epithelial cell lineage using the dissociated prostate cell regeneration system. We will (i) isolate individual lineages and test their regenerative capacity, and (ii) permanently mark individual lineages and determine the lineage status of their progeny. These can be achieved by creating a prostate basal cell-specific green fluorescence protein-marked transgenic mouse model or through the Cre-loxp marking system regulated by prostate lineage-specific promoters. (B) We will continue to screen the expression of surface antigens in prostate, fractionate prostate cells using surface antigens and identify the fraction(s) with enriched stem cell activity using the regeneration system. (2) Identify the prostate cancer-initiating cells. (A) We will evaluate the susceptibility of basal cells and luminal cells to oncogenic transformation. We will induce Pten deletion in individual lineages by infecting prostate epithelial cells from the PTENIoxp/loxp transgenic mouse model with lentivirus that express the Cre recombinase regulated by lineage-specific promoters. Infected cells will be tested in the regeneration system to determine which cell lineage(s) have been transformed. (B) We will determine the susceptibility of each cell population fractionated in Aim1 B to malignant transformation induced by single or a combination of oncogenic stimuli. Cell fractions from the wild type and P53-/- mice that represent stem cells, short-term progenitor cells and terminally differentiated cells will be infected with lentivirus that mediate distinct oncogenic signals, such as myc, inactivation of the pRB family proteins, perturbations in the PTEN-AKT signaling pathway and others. The infected cells will be microinjected into immunodeficient host mouse prostate or tested in the regeneration system to determine their capacity to initiate cancer.